By dipole-dipole interaction, FRET measures the distance between fluorescence molecules. Recently, smFRET has become a well-established method for studying biomolecular conformations with a robust distance quantification [1]}. We chose to use the ensemble trFRET method for several reasons. While smFRET has a limitation of detecting distances larger than 30 Å[2]}, using trFRET with smaller dyes can decrease the distance resolution down to \(>\)  5 Å. This enables the study of relatively small segments in the polypeptide chains. Furthermore, while it has been shown that smFRET dyes can promote collapse [3]}, [4]}, trFRET benefits from the lesser biased structural characterization by using small dyes such as natural amino acid (e.g., Tryptophan) [5]}. Moreover, while smFRET triumphs with direct observation to the ensemble sub-population it is less sensitive to the distance distributions' width as evaluated by trFRET [6]}, [7]}.
